The goal of this renewal application (for years 18 to 22) is to define the function of the recently identified 60 kDa albumin-binding glycoprotein (gp60) on the endothelial cell membrane in mediating the transendothelial flux of albumin. We have purified gp60 from bovine pulmonary microvessel endothelial cell (BPMVEC) membranes and lungs. Cross-linking of gp60 using an anti-gp60 antibody (Ab) and a secondary Ab to activate gp60 increased the endocytosis and permeability of 125I-labeled albumin 2 to 3 fold in endothelial monolayers and pulmonary microvessels of rats. Gp60 activation also increased the uptake and transport of horseradish peroxidase (a fluid phase marker). Gp60 was shown to co-localize with caveolin-1, the principal structural protein of caveolae in endothelial cell membrane gp60 activates the tyrosine kinase signaling pathway that mediates the internalization and transcytosis of albumin. The proposed studies have the following aims: (1) to identify the albumin binding domains of gp60 and the mechanism of interaction of albumin with gp60 that activates endocytosis, (2) to determine in endothelial monolayers and pulmonary microvessels the function of gp60 activation in mediating transcytosis of albumin and fluid phase macromolecules, 93) to study interaction of gp60 with caveolin-1, and activation of tyrosine kinase signaling pathway in regulating albumin endocytosis and transcytosis in endothelial cells, and 94) to determine the function of gp60 in mediating albumin transcytosis by gp60 cloning and its expression. With the achievement of these aims, we will be able to provide new insights into this important mechanism of albumin permeability in pulmonary vascular endothelial cells.